角蛋白45
Feather keratin hydrolysis by a Vibrio sp.strain kr2 S.Sangali and A.Brandelli
Departamento de Cie?ncia de Alimentos,ICTA,Universidade Federal do Rio Grande do Sul,Porto Alegre,Brazil
336/04/00:received19April2000,revised7June2000and accepted19June2000
S.S A N G A L I A N D A.B R A N D E L L I.2000.The aim of the study was to characterize feather-
degrading bacteria isolated from poultry industry waste.A Vibrio sp.strain kr2producing a
high keratinolytic activity when cultured on native feather-containing broth was isolated.
The bacterium grew with an optimum at pH6á0and30 C,where maximum feather-
degrading activity was also observed.Keratinase production was similar at both25and30
C,while the maximum concentration of soluble protein was reached at30 C.Reduction of
disulphide bridges was also observed,increasing with cultivation time.The keratinase of
strain kr2was active on azokeratin,azocasein,benzoyl-arginine-p-nitroanilide and Ala-Ala-
p-nitroanilide as substrates.The amino acid composition of the feather hydrolysate was
determined,presenting similarities with that reported for feather lysate,feather meal and
raw feathers.A novel feather-degrading bacterium was isolated and characterized,showing
high keratinolytic https://www.360docs.net/doc/e510111737.html,plete feather degradation was achieved during cultivation.
Strain kr2shows potential for use for biotechnological processes involving keratin
hydrolysis.
INTRODUCTION
Feathers,which are almost pure keratin protein(90%or more),are produced in large amounts as a waste by-pro-duct at poultry-processing https://www.360docs.net/doc/e510111737.html,lions of tons of feath-ers are produced annually worldwide and represent a potential alternative to more expensive dietary ingredients for animal feed.However,the current processes to obtain feather meal are expensive and also destroy certain amino acids,yielding a product with poor digestibility and vari-able nutrient quality(Wang and Parsons1997).Indeed,it is recognized that digestive enzymes,such as trypsin and pepsin,digest feather protein poorly.
The nutritional de?ciency of native feather protein derives from the composition and molecular con?gurations of constituent amino acids that provide,in essence,struc-tural rigidity for the feathers'role.The amino acid compo-sition of keratins from different avian species is very similar(O'Donnell and Inglis1974),as observed for differ-ent tissues of chicken feather(Harrap and Woods1964). The tight packing of the keratin chain in the a-helix(a-keratin)or b-sheet(b-keratin)into a supercoiled polypep-tide chain results in mechanical stability and resistance to proteolysis(Parry and North1998).In addition,cross-link-ing of protein chains by cysteine bridges confers high mechanical stability and resistance to proteolytic degrada-tion of keratins.The current investigation has been focused on proteolytic enzymes;however,reduction of cysteine bridges may have a signi?cant inˉuence on keratin degrada-tion(Noval and Nickerson1959;Kunert and Stransky 1988).
Keratinolytic enzymes may have important uses in bio-technological processes involving keratin-containing wastes from poultry and leather industries through the develop-ment of non-polluting processes(Shih1993;Onifade et al. 1998).These enzymes are produced by some species of Bacillus(Williams et al.1990;Atalo and Gashe1993),some actinomycetes(Noval and Nickerson1959;Boèckle et al. 1995)and other fungi(Kushwaha1983;Santos et al.1996). The use of keratinase to nutritionally upgrade feather meal has been described.A comparable growth rate was observed between chickens fed isolated soybean and those fed feather meal fermented with Streptomyces fradiae plus methionine supplementation(Elmayergi and Smith1971). The utilization of a Bacillus licheniformis feather lysate with amino acid supplementation produced a growth curve iden-tical to that of soybean meal(Williams et al.1991).The use of crude keratinase signi?cantly increased the amino acid digestibility of raw feathers and commercial feather meal(Lee et al.1991).
Searching for keratinolytic micro-organisms of industrial bene?t,an aerobic,Gram-negative,non-spore-forming, rod-shaped bacterium was isolated from industrial poultry waste at Porto Alegre,Brazil.This report describes the iso-
Correspondence to:A.Brandelli,ICTA±UFRGS,Av.Bento Gonc?alves9500, 91501±970Porto Alegre,Brazil(e-mail:abrand@vortex.ufrgs.br). Journal of Applied Microbiology2000,89,735à743
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lation of,identi?cation of,growth of and keratinase pro-duction by this feather-degrading isolate.
MATERIALS AND METHODS
Reagents
Azocasein,benzoyl-arginine-p-nitroanilide(BAPNA)and Ala-Ala-p-nitroanilide(APA)were from Sigma Chemical Co.(St.Louis,USA).Other reagents were from Merck (Darmstadt,Germany).
Isolation of keratinolytic micro-organism
Feathers were collected from several sites within a local poultry industry.Feathers wereˉooded in phosphate-buf-fered saline(PBS)and the suspension used to streak feather meal agar plates(15g là1feather meal,0á5g là1 NaCl,0á3g là1K2HPO4,0á4g là1KH2PO4and15g là1 agar)which were incubated at37 C for1±5d.Single colo-nies were isolated and screened for their ability to hydro-lyse keratin in feather meal agar plates.Colonies producing clearing zones in this medium were selected for further work.
Growth determination
The isolate was cultivated for72h in whole feather med-ium(15g là1whole feathers,0á5g là1NaCl,0á3g là1 K2HPO4,0á4g là1KH2PO4),from a106cfu mlà1culture. Cultures were grown in500-mlˉasks containing100ml medium,incubated in an orbital shaker at180rev minà1. Bacterial growth was monitored by measuring the colony-forming units(cfu mlà1),essentially as described by Miles and Misra(1938).The bacterial suspension was diluted to 10à8in PBS,samples were homogenized and then loaded (20m l)in triplicate onto nutrient agar plates.Plates were incubated for24h at30 C and counts done among30±100 colonies.
Taxonomical studies
The morphological,cultural and physiological characteris-tics of the isolated bacterium were compared with data from Bergey's Manual of Systematic Bacteriology(Brenner 1984).
Enzyme production
The organism was cultivated for72h in whole feather medium,from a106cfu mlà1culture.Samples were centri-fuged at10000g for5min and the supernatantˉuid used as a crude enzyme preparation.To study cell-associated enzyme,the pellet was washed with50mmol là1Tris buf-fer,pH8á0,and centrifuged at10000g for5min.The resulting pellet was resuspended in the same buffer and cell lysis was carried out by three cycles of10s in an ultra-sonic processor at20000Hz.Keratinase activity was then determined in the cell lysate and washing buffer.
Enzyme assay
Keratinase activity was assayed with azokeratin as a sub-strate by the following method.The reaction mixture con-tained100m l enzyme preparation and800m l2g là1 azokeratin in50mmol là1Tris buffer,pH8á0.The mixture was incubated for15min at50 C and the reaction then stopped by the addition of trichloroacetic acid to a?nal concentration of100g là1.After centrifugation at10000g for5min,the absorbance of the supernatantˉuid was determined at440nm.One unit of enzyme activity was the amount of enzyme that caused a change of absorbance of 0á01at440nm for15min at50 C.Azokeratin was synthe-sized as described elsewhere(Tomarelli et al.1949).A similar protocol was used to determine enzyme activity on azocasein.
Enzyme activity on synthetic substrates
Protease activity on the synthetic substrates BAPNA and APA was determined.The reaction mixture contained100 m l enzyme preparation and400m l10mmol là1substrate in 50mmol là1Tris buffer,pH8á0.The mixture was incu-bated for15min at50 C and the reaction then stopped by adding acetic acid to a?nal concentration of300g là1. After centrifugation at10000g for5min the absorbance of the supernatantˉuid was determined at405nm.One unit represented the amount of enzyme necessary to catalyse the production of1m mol p-nitroanilide minà1at50 C.
Determination of protein concentration
A cell-free supernatantˉuid of each feather culture was used for the determination of soluble protein by the Folin phenol reagent method(Lowry et al.1951)with bovine serum albumin as standard.
Determination of thiol formation
The determination of free thiol groups was carried out essentially as described elsewhere(Sullivan et al.1942).To 1ml sample were added0á2ml NH4OH,1ml0á5g là1 NaCN and1ml water.The mixture was incubated for20 min at25 C and then0á2ml0á5g là1sodium nitroprusside was added.Absorbance at530nm was determined within2 min.
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Feather hydrolysate preparation
Strain kr2was grown on whole feather broth for72h and the supernatantˉuid of the culture was obtained by centri-fugation at10000g for5min.The supernatantˉuid was dried at135±139 C and2ml minà1in a spray dryer (Buèchi Labortechnik,Flawil,Switzerland).This material was analysed for protein,ash and moisture by standard procedures(Association of Of?cial Analytical Chemists 1984).
Amino acid analysis
Amino acid analysis was performed using a Sequential Multi-Sample Amino Acid Analyser(TM S2;Technicon). The amino acids were obtained by peptide hydrolysis with 6mol l-1HCl at110 C for24h and puri?ed with Amberlite IR-120(Sigma,St Louis,USA)before analysis.
RESULTS
Isolation and identification of keratinase-producing strains
Screening for feather-degrading micro-organisms was car-ried out from feathers in decomposition.Five distinct col-ony morphologies were observed when streaked onto feather meal agar plates.The strain designation kr1,kr2, kr3,kr4and kr5was proposed to indicate the isolation of
the keratinolytic strains.Isolates kr1and kr5were Gram-positive rods,kr2and kr3were Gram-negative rods and kr4were Gram-negative cocci.Three of them produced clearing zones in feather meal agar(Fig.1)and kr2demon-strated pronounced growth in whole feather medium.This isolate was selected for identi?cation and for its adaptation to feather degradation.Cells of the isolate were grown on whole feathers and transferred at frequent intervals to the basal medium containing whole feathers.The isolate was able to complete the disintegration of whole feathers.
The results of taxonomical studies on the isolated strain kr2are summarized in Table1.The identi?cation of this bacterial isolate was based on cell morphology,colony mor-phology,growth characteristics and several biochemical tests.Microscopic observation of the isolate showed a straight rod and,after prolonged incubation,slightly curved rods were observed.The bacterium grew aerobically and was strongly sensitive to Vibrio static agent O/129. The Vibrio static agent plus enzyme activities were key tests to differentiate genera within the Vibrionaceae family (Altwegg1994;Bottone and Janda1994).Organisms belonging to the genera Vibrio and Plesiomonas are sensitive to Vibrio static agent O/129;however,organisms belonging to Plesiomonas lack exoenzymes and are often positive for arginine dihydrolase,lysine decarboxylase and ornithine decarboxylase.According to Bergey's Manual of Systematic Bacteriology(Brenner1984),these characteristics indicated that the isolate was of the family Vibrionaceae,genus Vibrio.Strain kr2grew very slowly in sheep blood agar where no b-haemolysis was observed.
Optimal growth conditions
Growth of the newly isolated strain occurred between pH 6á0and8á0and between25and37 C,with an optimum at pH6á0and30 C.Growth was detected between5and50g là1NaCl.The effect of temperature on bacterial growth is shown in Fig.2a.Similar curves were observed at both25 and30 C.When incubation was at37 C,minor growth was observed and no complete feather degradation was achieved.Different concentrations of NaCl(up to50g là1) or addition of5g là1NH4Cl caused no effect on the growth of strain kr2(Fig.2b).Different pH values were tested for bacterial growth and the results are shown in Fig.3.The bacterium grew similarly at initial pHs of6á0or 7á0,whereas minor growth was observed at pH8á0.When the initial pH was6á0or7á0,these values increased to8á0 at the end of
incubation.
Fig.1Production of clearing zones in feather meal agar by keratinolytic bacteria.Micro-organisms were inoculated by stick and plates incubated at30 C for5d
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Table 1Morphological,cultural and physiological characteristics of keratinase-producing bacterial strain kr2
Morphological characteristics Form Rods
Size
0á5?1±2m m Gram stain Negative
Spore
Non-sporulating
Cultural characteristics
Feather meal agar colonies Yellowish-pink colour,circular,smooth,convex,undulate,moist Physiological characteristics Catalase Positive Oxidase Positive O-F test Negative V-P test Negative Citrate
Negative Nitrate reduction Positive Gelatin liquefaction Positive Starch hydrolysis Positive Lipase Positive DNAse
Positive Lysine decarboxylase Negative Ornithine decarboxylase Negative Arginine dihydrolase Negative O/129Vibrio static agent Sensitive
Triple sugar iron agar Alkaline slant/alkaline butt/no H 2S production Motility
Negative
Fig.2Effect of temperature and media composition on growth of vibrio strain kr2.(a)Growth of strain kr2monitored at .
,25,&,30and ~,37 C.(b)Growth of strain kr2in whole feather medium containing .
,50g l à1NaCl or &,5g l à1NH 4Cl.Each point represents
the mean of three independent experiments
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Growth substrates
The new isolate was able to use starch,sucrose,glucose,fructose,pectin,casein,peptone and succinate as growth substrates.Casein and peptone,as both C and N source,were the preferred substrates,con?rming the proteolytic character of strain kr2.Isolate kr2was not able to utilize carboxymethyl cellulose,cellulose or Tween 80.
Production of keratinase and soluble protein
The effect of pH and temperature on the production of keratinase and soluble protein was investigated.The maxi-mum enzyme production was observed by growing the organism at 30 C,although similar levels were observed at 25 C (Table 2).The highest amount of soluble protein was also observed at 30 C.Similar levels of protein were reached at both pH 6á0and 8á0,while keratinase production was higher at pH 6á0.At this last initial pH more ef?cient feather degradation was observed.
The activity of keratinase was followed during cultiva-tion of strain kr2in whole feather medium at 30 C.The highest amount of keratinolytic activity produced was found in the supernatant ˉuid of the culture medium (Fig.4).Keratinase reached a maximum activity at 32h,coincid-ing with the end of the exponential phase,then decreasing and increasing again at 72h.The keratinolytic activity asso-ciated to cell lysate increased to a maximum at 32h,remaining at this value until the end of incubation.
Reduction of disulphide bonds
The high stability of keratin proteins against mechanical and enzymatic attack is due,at least in part,to the occur-rence of cysteine bridges.The reduction of disulphide bonds by strain kr2was investigated.Thiol formation increased with cultivation time (Fig.5),reaching a maxi-mum at 72h.The rate of thiol formation was maximal dur-ing the exponential
phase.
Fig.3Effect of pH on growth of vibrio strain kr2.(a)pH during cultivation of strain kr2at an initial pH of .
,6á0and &,7á0.(b)Growth of strain kr2at an initial pH of .
,6á0,&,7á0and ~,8á0.Each point represents the mean of three independent experiments
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Substrate specificity
The keratinase preparation was tested on azokeratin,azoca-sein,BAPNA and APA as substrates.The enzyme was active on all substrates tested,showing about twofold
higher activity on azocasein than azokeratin (Fig.6).The enzyme was also active on bovine serum albumin as sub-strate.
Amino acid composition
The amino acid composition of kr2feather hydrolysate is summarized in Table 3.The hydrolysate is rich in serine,leucine,alanine and glutamate residues and contains minor amounts of histidine and methionine.The percentage of amino acids involved in the proteolysis breakdown by tryp-sin-like (Lys and Arg),chymotrypsin-like (Tyr,Phe,Leu and Met)and elastase-like (Ala,Gly and Ser)serine protei-nases were 10á8,20á1and 21á7%,respectively.
DISCUSSION
A bacterium was isolated from a poultry-processing plant,presenting feather-degrading properties.Strain kr2was tentatively identi?ed as Vibrio sp.and may have adapted to utilize feathers as substrate.The isolate showed optimum growth at mesophilic temperatures,as expected for a micro-organism of environmental origin.Other previously described keratinolytic bacteria generally have optimum growth and feather degradation activity at high tempera-
Table 2Effect of temperature and pH on keratinase and soluble
protein production by kr2strain*
Keratinase (units ml à1)
Protein (g l à1)Temperature ( C)2529 51á8 0á3
3032 42á5 0á63712 21á4 0á242ND ND Initial pH 6á031 62á6 0á57á024 42á0 0á58á027 6
2á8 0á3
*Determinations were carried out after 72h growth in whole feather medium and are the mean S .E .M .of three independent experiments.ND,Not
detected.
Fig.4Distribution of keratinase activity during growth of vibrio
strain kr2in whole feather medium.Extracellular (.,culture ?ltrate plus washing buffer)and intracellular (&,cell lysate)
activities were measured using azokeratin as substrate.Each point represents the mean of three independent
experiments
Fig.5Formation of extracellular thiol groups during growth of
vibrio strain kr2in whole feather medium.Each point represents the mean of three independent experiments
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tures (Williams et al .1990;Atalo and Gashe 1993).Until now,feather degradation by bacteria has only been described for Bacillus spp.(Williams et al .1990;Atalo and Gashe 1993;Zaghloul et al .1998).
The increase in pH during cultivation is an important characteristic accompanying keratin hydrolysis and the ker-atinolytic potential of https://www.360docs.net/doc/e510111737.html,anisms with a higher keratinolytic activity turn the media more alkaline,in comparison with other organisms exhibiting lower kera-tinolytic activity (Kaul and Sumbali 1997).This observa-tion is based on the fact that keratin degradation involves deamination reactions,which result in an increase in pH.Isolate kr2caused a signi?cant increase in pH during culti-vation on raw feathers and was able to complete feather degradation,indicating its strong keratinolytic character.The production of keratinolytic enzymes by the isolate was maximal at 30 C and pH 6á0,whereas protease pro-duction by other feather-degrading bacteria normally occurs at higher pH and temperature (Williams et al .1990;Atalo and Gashe 1993).Strain kr2produced an extracellu-lar keratinase whose levels were variable during cultivation on whole feathers.This pattern may suggest that the enzyme is inducible,i.e.substrate levels in the extracellular milieu regulate its secretion.These results resemble those observed for cultures of streptomyces (Shama and Berwick
1991;Bo
èckle et al .1995),trichophyton (Grzywnowicz et al .1989)and bacillus (Atalo and Gashe 1993)on keratin sub-strates.
Thiol formation by strain kr2suggests the presence of disulphide reductase activity.Reduction of disulphide bridges was also observed for S.pactum grown on feather (Bo èckle and Mu èller 1997)and S.fradiae on wool (Shama and Berwick 1991).In addition,the use of reducing agents to enhance keratin degradation by keratinases has been
described (Bo
èckle et al .1995;Letourneau et al .1998).Thus the screening for keratinolytic micro-organisms should also lead to the isolation of organisms possessing disulphide reductases.
Vibrio strains of environmental origin produce proteases of potential technological interest,usually serine proteases (Kwon et al .1995;Marcello et al .1996).The main proteo-lytic activity of keratinases is normally associated with ser-ine proteinase activity (Bo
èckle et al .1995;Lin et al .1995;Lin et al .1999).Strain kr2possessed serine protease activ-ity,as evidenced by hydrolysis of the substrate BAPNA.The enzyme preparation of kr2was also active on APA,indicating the presence of arylamidase activity,and azoca-sein,indicating its endoproteolytic character.These data suggest a broad substrate speci?city of kr2keratinase.
The amino acid composition of the kr2hydrolysate resembles that reported for a B.licheniformis feather lysate (Williams et al .1991).Serine and leucine have also been described as main residues in raw feathers,feather
meal
Fig.6Activity of vibrio kr2keratinase on different substrates.
Proteolytic activity was tested on azokeratin,azocasein,benzoyl-arginine-p -nitroanilide (BAPNA)and Ala-Ala-p -nitroanilide
(APA)as described in the Materials and Methods.Bars represent the mean S .E .M .of three independent experiments
Table 3Amino acid composition of chicken feather hydrolysate*
Amino acid Mol percentage Ala 5á42Arg 8á43Asp {5á78Cys 6á51Glu {9á22Gly 5á96His 0á93Ile 6á28Leu 9á69Lys 2á41Met 1á70Phe 5á42Pro 5á28Ser 10á83Thr 3á66Tyr 3á29Val
8á56
*Values are the average of triplicate samples.Dry matter,906á5mg g à1;crude protein,651á8mg g à1;ash,158á8mg g à1.{Value for aspartic acid and asparagine.{Value for glutamic acid and glutamine.
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(Papadopoulos et al.1986;Williams et al.1991)and protein B4isolated from feather barbs(Arai et al.1983).Feather protein has been considered as an excellent source of meta-bolizable protein(Klemersrud et al.1998)and bacterial feather lysate has similar nutritional features to soybean meal(Williams et al.1991),indicating the potential use of kr2to produce feed protein.
This new isolated strain possesses high keratinolytic activity and is very effective in feather degradation,pre-senting potential use for biotechnological processes invol-ving keratin hydrolysis.
ACKNOWLEDGEMENTS
This work was supported by FAPERGS and FAURGS. S.S.has received an M.Sc.fellowship from CAPES and A.B.is a Research Fellow of CNPq,Brazil.
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=2000The Society for Applied Microbiology,Journal of Applied Microbiology,89,735à743